1. Field of the Invention
This invention relates to a method for the isolation of nucleic acids using a material with an unmodified silica surface, such as magnetic glass particles, and subsequent amplification of a target nucleic acid in the presence of the material with an unmodified silica surface. The method is preferably carried out as an automated process preferably in a high throughput format. The method is preferably used in diagnostics.
2. Background Art
Many biological substances, especially nucleic acids, present special challenges in terms of isolating them from their natural environment. On the one hand, they are often present in very small concentrations and, on the other hand, they are often found in the presence of many other solid and dissolved substances e.g. after lysis of cells. This makes them difficult to isolate or to measure, in particular in biospecific assays which allow the detection of specific analytes, e.g. nucleic acids, or specific analyte properties and play a major role in the field of diagnostics and bioanalytics in research and development.
Before a biological substance or compound, for example a nucleic acid, can be analyzed in a biospecific assay or used for other processes, it often must be isolated or purified from biological samples containing complex mixtures of different components as e.g. proteinaceous and non-proteinaceous components. Frequently, the biological substance is contained in a bacterial cell, a fungal cell, a viral particle, or the cell of a more complex organism, such as a human blood cell or a plant cell. The biological substance to be analyzed is also frequently called substance of interest or target substance. Frequently, the target substance is a nucleic acid which is therefore called a target nucleic acid.
To release the contents of said cells or particles, they may be treated with enzymes or with chemicals to dissolve, degrade or denature the cell walls and/or cell membranes. This process is commonly referred to as lysis. The resulting solution containing such lysed material is referred to as lysate. A problem often encountered during the lysis is that enzymes degrading the substance of interest, e.g. deoxyribonucleases or ribonucleases degrading nucleic acids, come into contact with the substance of interest during lysis. These degrading enzymes may be present outside the cells or may have been spatially separated in different cellular compartments before the lysis. Other components released during this process might include, for example, endotoxins belonging to the family of lipopolysaccharides which are toxic to cells and can cause problems for products intended to be used in human or animal therapy. There are a variety of means to tackle this problem mentioned-above. It is common to use chaotropic agents as e.g. guanidinium salts or anionic, cationic, zwitterionic or non-ionic detergents when nucleic acids are intended to be set free. It is also an advantage to use proteases, e.g. proteinase K, which rapidly degrade these enzymes or unwanted proteins. However, this may produce another problem as the said substances or enzymes can interfere with reagents or components in subsequent steps.
If the substances of interest are nucleic acids, they are normally extracted and thus separated from the complex lysis mixtures before they are used in an assay. There are several methods for the extraction of nucleic acids as sequence-dependent or biospecific methods (affinity chromatography, hybridisation to immobilised probes) or sequence-independent or physico-chemical methods (liquid-liquid extraction with e.g. phenol-chloroform, precipitation with e.g. pure ethanol, extraction with filter paper, extraction with micelle-forming agents as cetyl-trimethyl-ammonium-bromide, binding to immobilised, intercalating dyes, e.g. acridine derivatives, adsorption to silica gel or diatomic earths, adsorption to magnetic glass particles (MGPs) or organo silane particles under chaotropic conditions. Extraction using solid phases usually comprises the steps of adding the lysate to the solid phase under conditions allowing binding of the substance of interest to the solid phase, removal of the remainder of the lysate from the solid phase bound substance and subsequent release of the substance of interest from the solid phase into a liquid eluate (sometimes called elution). The result of the extraction process is usually a solution containing the substance of interest in dissolved state. Particularly interesting for extraction purposes is the adsorption of nucleic acids to a glass surface, in particular the glass surfaces of MGPs. Many procedures for isolating nucleic acids from their natural environment have been proposed in recent years by the use of their binding behavior to glass surfaces.
After the extraction step, the solution containing the substance of interest, e.g. the nucleic acid, is analyzed in a biospecific assay to show whether the substance of interest was present in the original sample. Examples for biospecific assays are hybridization assays for nucleic acids, immuno assays or receptor-ligand assays for proteins. Hybridization assays use the specific base-pairing for the molecular detection of nucleic acid analytes, for example, RNA or DNA. Hence, for example, oligonucleotide probes with sequence of a length of about 18 to about 20 nucleotides may enable the specific recognition of a selected complementary sequence, for example, in the human genome. Another assay which entails the selective binding of two oligonucleotide primers is the polymerase chain reaction (PCR) described in U.S. Pat. No. 4,683,195. This method allows the selective amplification of a specific nucleic acid region to detectable levels by a thermostable polymerase in the presence of desoxynucleotide triphosphates in several cycles. Afterwards the nucleic acid is detected by means known to the person skilled in the art. Other methods, such as the TAQMAN® assay disclosed in WO92/02638, allow the simultaneous amplification and detection of a nucleic acid of interest.
Normally the lysis, extraction and amplification steps are performed consecutively while a variety of different operations have to be carried out, e.g. removal of only protein containing phases, elution of the nucleic acids from the carried used for extraction and removal of the carrier, transfer of liquids into fresh tubes and so on. New methods of preparing nucleic acid samples are needed to improve the efficiency and/or sensitivity of, for example, nucleic acid detection methods.